Spatiotemporally Resolved Mapping of Nuclear-associated Protein Through Photocatalytic Proximity Labeling

Abstract

Spatiotemporal profiling of nuclear-associated proteomes is crucial for elucidating disease mechanisms, identifying key therapeutic targets, and guiding the design of effective drugs. Currently, proximity labeling (PL) using genetically transfected enzymes or photocatalyst-based probes has emerged as a powerful tool for proteomic mapping. However, these approaches are limited by their incompatibility with hard-to-transfect cells and primary tissues, as well as by the lack of efficient nucleus-targeting strategies. In this study, we developed a photocatalytic PL strategy (Pc-PL) that enables efficient enrichment of nuclear-associated proteins by combining a nucleus-targeted photosensitizer (NCP) with photocatalysis-mediated reactive biotin labeling. Compared with traditional photocatalysts such as chlorin e6 and rose Bengal, NCP exhibited superior nuclear accumulation across various cell types. Cellular experiments confirmed that NCP-mediated photoactivation precisely localized biotin labeling within the nucleus, enabling selective enrichment of nuclear proteins via subsequent streptavidin-based magnetic capture. Coupling Pc-PL with quantitative mass spectrometry enabled highresolution mapping of nuclear proteomes and led to the discovery of previously unrecognized senescence-associated regulators, including TMPO. Collectively, these findings establish Pc-PL as an innovative and versatile tool for highresolution nuclear proteomics, offering broad potential for target discovery and drug development.

Keywords

drug development / photosensitizer / proteomics / proximity labeling / senescence

References

[1] D. E. Scott, A. R. Bayly, C. Abell, and J. Skidmore, “Small Molecules, Big Targets: Drug Discovery Faces the Protein-protein Interaction Challenge,” Nature Reviews Drug Discovery 15 (2016): 533-550.

[2] D. V. Fyodorov, B. R. Zhou, A. I. Skoultchi, and Y. Bai, “Emerging Roles of Linker Histones in Regulating Chromatin Structure and Function,” Nature Reviews Molecular Cell Biology 19 (2018): 192-206.

[3] P. M. Davidson and B. Cadot, “Actin on and Around the Nucleus,” Trends in Cell Biology 31 (2021): 211-223.

[4] H. T. J. Gilbert and J. Swift, “The Consequences of Ageing, Progeroid Syndromes and Cellular Senescence on Mechanotransduction and the Nucleus,” Experimental Cell Research 378 (2019): 98-103.

[5] W. Qin, K. F. Cho, P. E. Cavanagh, and A. Y. Ting, “Deciphering Molecular Interactions by Proximity Labeling,” Nature Methods 18 (2021): 133-143.

[6] H. W. Rhee, P. Zou, N. D. Udeshi, et al., “Proteomic Mapping of Mitochondria in Living Cells via Spatially Restricted Enzymatic Tagging,” Science 339 (2013): 1328-1331.

[7] S. S. Lam, J. D. Martell, K. J. Kamer, et al., “Directed Evolution of APEX2 for Electron Microscopy and Proximity Labeling,” Nature Methods 12 (2015): 51-54.

[8] T. C. Branon, J. A. Bosch, A. D. Sanchez, et al., “Efficient Proximity Labeling in Living Cells and Organisms With TurboID,” Nature Biotechnology 36 (2018): 880-887.

[9] M. S. Al-Dosari and X. Gao, “Nonviral Gene Delivery: Principle, Limitations, and Recent Progress,” The AAPS Journal 11 (2009): 671-681.

[10] Z. Liu, F. Guo, Y. Zhu, et al., “Bioorthogonal Photocatalytic Proximity Labeling in Primary Living Samples,” Nature Communications 15 (2024): 2712.

[11] N. Zhou, Y. Zhang, P. R. Chen, and X. Fan, “Time-resolved Photocatalytic Proximity Labeling Uncovers ER Proteome Dynamics Underlying UPR-to-apoptosis Transition,” Proceedings of the National Academy of Sciences of the United States of America 122 (2025): e2503115122..

[12] Z. Huang, Z. Liu, X. Xie, et al., “Bioorthogonal Photocatalytic Decaging-Enabled Mitochondrial Proteomics,” Journal of the American Chemical Society 143 (2021): 18714-18720.

[13] C. P. Seath, A. J. Burton, X. Sun, et al., “Tracking Chromatin state Changes Using Nanoscale Photo-proximity Labelling,” Nature 616 (2023): 574-580.

[14] R. Ma, Y. Zhang, J. Zhang, et al., “Targeting Pericentric Non-consecutive Motifs for Heterochromatin Initiation,” Nature 631 (2024): 678-685.

[15] L. Chen, N. Li, M. Zhang, et al., “APEX2-based Proximity Labeling of Atox1 Identifies CRIP2 as a Nuclear Copper-binding Protein That Regulates Autophagy Activation,” Angewandte Chemie International Edition 60 (2021): 25346-25355.

[16] P. Wang, Q. Bai, X. Liu, et al., “Nucleus-Targeting Photosensitizers Enhance Neutrophil Extracellular Traps for Efficient Eradication of Multidrug-Resistant Bacterial Infections,” Advanced Materials 36 (2024): e2400304.

[17] Y. Zhou, G. Wang, P. Wang, et al., “Expanding APEX2 Substrates for Proximity-Dependent Labeling of Nucleic Acids and Proteins in Living Cells,” Angewandte Chemie International Edition 58 (2019): 11763-11767.

[18] W. Qin, S. A. Myers, D. K. Carey, S. A. Carr, and A. Y. Ting, “Spatiotemporally-resolved Mapping of RNA Binding Proteins via Functional Proximity Labeling Reveals a Mitochondrial mRNA Anchor Promoting Stress Recovery,” Nature Communications 12 (2021): 4980.

[19] F. Zheng, C. Yu, X. Zhou, and P. Zou, “Genetically Encoded Photocatalytic Protein Labeling Enables Spatially-resolved Profiling of Intracellular Proteome,” Nature Communications 14 (2023): 2978.

[20] T. Yasuhara, Y. H. Xing, N. C. Bauer, et al., “Condensates Induced by Transcription Inhibition Localize Active Chromatin to Nucleoli,” Molecular Cell 82 (2022): 2738-2753.e6.

[21] E. Buratti and F. E. Baralle, “TDP-43: Gumming up Neurons Through Protein-protein and Protein-RNA Interactions,” Trends in Biochemical Sciences 37 (2012): 237-247.

[22] C. Wen, L. Jiang, P. Pan, et al., “GRSF1 deficiency Attenuates Mitochondrial Function in Aging Granulosa Cells,” Reproduction 168 (2024), e240015.

[23] R. Di Micco, V. Krizhanovsky, D. Baker, and F. d'Adda di Fagagna, “Cellular Senescence in Ageing: From Mechanisms to Therapeutic Opportunities,” Nature Reviews Molecular Cell Biology 22 (2021): 75-95.

[24] I. Marin, O. Boix, A. Garcia-Garijo, et al., “Cellular Senescence is Immunogenic and Promotes Antitumor Immunity,” Cancer Discovery 13 (2023): 410-431.

[25] J. Hong, S. Min, G. Yoon, and S. B. Lim, “SRSF7 Downregulation Induces Cellular Senescence Through Generation of MDM2 Variants,” Aging 15 (2023): 14591-14606.

[26] J. Wang, D. Xia, Y. Lin, et al., “Oxidative Stress-induced circKIF18A Downregulation Impairs MCM7-mediated Anti-senescence in Intervertebral Disc Degeneration,” Experimental & Molecular Medicine 54 (2022): 285-297.

[27] S. Sikder, S. Baek, T. McNeil, and Y. Dalal, “Centromere Inactivation During Aging Can be Rescued in Human Cells,” Molecular Cell 85 (2025): 692-707.e7.

[28] X. Du, X. Zhang, Z. Qi, et al., “HELLS Modulates the Stemness of Intrahepatic Cholangiocarcinoma Through Promoting Senescence-associated Secretory Phenotype,” Computational and Structural Biotechnology Journal 21 (2023): 5174-5185.

[29] M. H. Li, X. Jiang, Y. Jing, et al., “CRISPR-based Screening Pinpoints H2AZ1 as a Driver of Senescence in Human Mesenchymal Stem Cells,” Protein Cell 16 (2025): 293-299.

[30] A. Hanswillemenke, D. T. Hofacker, M. Sorgenfrei, et al., “Profiling the Interactome of Oligonucleotide Drugs by Proximity Biotinylation,” Nature Chemical Biology 20 (2024): 555-565.

[31] D. G. May, K. L. Scott, A. R. Campos, and K. J. Roux, “Comparative Application of BioID and TurboID for Protein-Proximity Biotinylation,” Cells 9 (2020): 1070.

[32] P. K. Liu, Z. Wang, and L. Li, “Recent Advances in Chemical Proteomics for Protein Profiling and Targeted Degradation,” Current Opinion in Chemical Biology 87 (2025): 102605.

RIGHTS & PERMISSIONS

2026 The Author(s). Aggregate published by SCUT, AIEI, and John Wiley & Sons Australia, Ltd.